Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts

Abstract

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

Keywords: autophagy; differentiation; mitochondria; mitophagy; myoblasts; myogenesis; myotubes.

Figure 1.

Autophagy is upregulated during myogenic differentiation of C2C12 myoblasts. C2C12 skeletal myoblasts were differentiated for 6 d in low serum differentiation media. (A) Phase contrast microscopy of differentiating C2C12s in growth medium (GM) or 1 d, 3 d, or 6 d PD. Scale bars: 100 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B normalized to ARHGDIA (**, P < 0.01; *****, P < 0.00001; Student t test; representative western blot is shown, n=3).

Figure 2.

Blocking autophagy prevents myogenic differentiation. C2C12 cells were pretreated with autophagy-inhibiting agents and were subsequently differentiated. (A, C, and E) Phase contrast microscopy of differentiating C2C12s pretreated with either siRNA targeting Atg5 (A), BAF (C), or siRNA targeting Sqstm1 prior to differentiation (E). Scale bars: 100 µm. (B, D, and F) Western blot analysis of whole cell lysates from siAtg5 (B), BAF (D), or siSqstm1 (F)-treated cells. GM, growth medium.

Figure 3.

Mitochondrial remodeling occurs during myogenic differentiation. Differentiating C2C12s were examined for alterations in mitochondrial networks. (A) Cells expressing mitochondria-targeted DsRed were differentiated and examined with fluorescence microscopy. Exposure times were individually adjusted to bring out detail. Scale bars: 20 µm. (B) Differentiating cells immunostained for OPA1 (red) and imaged via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (C) Western blot analysis of whole cell lysates from differentiating C2C12s. (D) Quantification of western blots in C normalized to ARHGDIA (*, P < 0.05; **, P < 0.01; Student t test; representative western blot is shown, n=3). GM, growth medium.

Figure 4.

Electron micrographs of differentiating C2C12s. Transmission electron microscopy was performed on differentiating C2C12s to examine alterations in mitochondrial populations. Insets are presented at higher magnification below each original image. Scale bars: 500 nm. GM, growth medium.

Figure 5.

Mitochondrial remodeling is impaired when autophagic flux is blocked. BAF-treated cells were examined to determine if autophagic flux was required for mitochondrial remodeling. (A) C2C12s were pretreated with BAF or vehicle and then stained for TOMM70A (green) and with Hoechst 33342 (blue) and imaged via fluorescence microscopy. Insets are presented at higher magnification below each original image. Exposure times were individually adjusted to bring out detail. Scale bars: 20 µm. (B) Cells in A were analyzed using Image J to measure mitochondrial pixel area/perimeter ratios of individual cells as a measure of network interconnectivity. (***, P<.001; *****, P<.00001; Student t test, n=10) (C) Western blot analysis of whole cell lysates from BAF-treated C2C12s. (D) Quantification of western blots in C. GM, growth medium.

Figure 6.

Electron micrographs of differentiating C2C12s treated with BAF. Transmission electron microscopy was performed on differentiating C2C12s treated with 100 nM BAF to examine alterations in mitochondrial populations. Insets are presented at higher magnification below each original image. Scale bars: 500 nm. GM, growth medium.

Figure 7.

Myogenic differentiation is accompanied by mitophagy and biogenesis. Differentiating C2C12s were analyzed for mitophagy and biogenesis markers. (A) Differentiating C2C12s immunostained for TOMM70A (green) and imaged via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B. Whole lysate blots were normalized to ARHGDIA. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student t test; representative western blot is shown, n=3). (D) Differentiating C2C12s were immunostained for PPARGC1A (red) as well as with Hoechst 33342 nuclear stain (blue) and examined via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (E) Quantification of the percentage of PPARGC1A⁺ nuclei over total nuclei (100 nuclei counted per condition). GM, growth medium.

Figure 8.

Assessment of mitochondrial turnover via MitoTimer. Mitochondrial turnover was examined in differentiating C2C12s expressing MitoTimer. (A) Fluorescent images of MitoTimer-expressing C2C12s throughout differentiation. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Red/green ratios based on images from A. Three fields were analyzed per time point. (C) PE/FITC ratios of differentiating MitoTimer-expressing C2C12s as measured by fluorescence-activated cell sorting. 50,000 events were analyzed per time point. GM, growth medium.

Figure 9.

Myotubes are populated with more tightly coupled mitochondria fortified with increased OXPHOS machinery. Undifferentiated C2C12 myoblasts and differentiated myotubes 6 d PD were examined for qualitative differences in mitochondria. (A) OCRs of isolated mitochondria from myoblasts (Blast Mito) or myotubes (Tube Mito). Palmitoyl carnitine was used as the substrate. Average coupled ratios are presented below. (B) OCRs of intact myoblasts or myotubes using palmitate as the substrate. (C) Western blot analysis detecting OXPHOS complex proteins in mitochondria isolated from myoblasts or myotubes. (D) Quantification of MT-CO1 western blot (*, P < 0.05; Student t test; representative western blot is shown, n=3). GM, growth medium.

Figure 10.

Schematic of mitochondrial remodeling during differentiation. (A) Myoblasts rely primarily on glycolysis and to a lesser extent glucose oxidation and contain sparsely-populated networks of mitochondria. (B) With a differentiation stimulus, mitochondrial fission protein DNM1L and mitophagy receptor protein SQSTM1 are upregulated, leading to mitochondrial fragmentation and mitophagy. (C) Following clearance of the myoblast mitochondria, new mitochondria are synthesized via PPARGC1A-mediated biogenesis. These new mitochondria which rely primarily on fatty acid oxidation, are more tightly-coupled and better equipped to perform OXPHOS. Dense mitochondrial networks are established via OPA1-mediated fusion.